Clinical and Molecular Analysis in Patients with Peutz-Jeghers Syndrome

Background/Aims: Peutz–Jeghers syndrome (PJS) is a rare hereditary disorder linked to increased cancer risk due to specific genetic variants in the STK11 gene. This study aimed to assess disease manifestations, genetic profiles, and genotype–phenotype correlations in PJS patients. Materials and Methods: Twenty patients from 14 families with PJS who were followed up at our clinic between 2011 and 2021 were included. Genetic susceptibility to hereditary cancers was assess–ed using targeted next-generation sequencing (NGS) and multiplex ligation-dependent probe amplification (MLPA) of the STK11 gene. Clinical data were also collected and analyzed in conjunction with the genetic findings. Results: Initial symptoms appeared around 18.9 years, predominantly abdominal pain and intussusception. Mucocutaneous lesions were found in 85%, and hamartomatous polyps in 90%. Dysplastic polyps were found in 4 patients, with 3 cases of malignancy. Next-generation sequencing identified 11 pathogenic and 3 likely pathogenic mutations, including 3 novel STK11 variants (LRG_319: c.598-8_601del, LRG_319: c.708_718del, and LRG_319: c.146_147del). Next-generation sequencing diagnostic rate was 78.5% (11/14), and the overall diagnostic rate with NGS and MLPA studies was 85.7% (12/14). Patients without STK11 mutations had later symptom onset and potentially lower cancer risk. Truncated mutations are associated with earlier symptoms and elevated cancer risk. Conclusion: This is the first PJS case series in Türkiye using the NGS and MLPA methods. It reports 3 novel mutations and emphasizes the genotype–phenotype relationship of PJS. With further studies, the genotype–phenotype relationship of STK11 variants will be better understood.


INTRODUCTION
2][3] Its hallmark features include hamartomatous polyps within the gastrointestinal tract, particularly the small intestine, and distinctive mucocutaneous pigmentation, often emerging during adolescence. 4agnosis of PJS relies on clinical presentation and family history, as outlined in Table 1. 5 This syndrome associated with an elevated risk of gastrointestinal and extragastrointestinal cancers, 6 with a reported lifetime risk of 55%-85%. 5enetic analysis contributes to diagnosis confirmation, clinical interpretation, and early risk detection.
Peutz-Jeghers syndrome arises from germline mutations in the STK11 tumor suppressor gene located on chromosome 19p13.3.This gene encodes a serin e/thr eonin e-pro tein kinase that regulates the mammalian rapamycin target pathway. 7According to the Human Gene Mutation Database (http: //www .hgmd.cf.a c.uk), over 400 pathogenic or likely pathogenic STK11 mutations have been recorded in individuals with PJS. 8 Despite extensive genotype-phenotype research, correlation between STK11 pathogenic variants and associated phenotypes remains debated. 9Amos et al 10 reported that individuals with truncating variants had similar onset ages for initial polyps, whereas those with missense variants exhibited later onset.Similarly, Salloch et al 11
[14] This study mainly aims to investigate the genetic etiology, establish genotype-phenotype correlations, and provide a comprehensive clinical and genetic profile of patients with PJS in a Turkish cohort.Utilizing a targeted next-generation sequencing (NGS) panel and multiplex ligationdependent probe amplification (MLPA) analysis, the study seeks to contribute insights into the relationship between STK11 gene variants and the clinical manifestations of PJS.

MATERIALS AND METHODS
This study was approved by the Dokuz Eylül University Non-Interventional Research Ethics Committee (No: 2021/20-44, Date: April 19, 2021).The study included patients followed at the Gastroenterology and Pediatric Gastroenterology Clinics at Dokuz Eylül University Faculty of Medicine from January 2011 to April 2021.Patients meeting at least 1 World Health Organization diagnostic criterion for PJS (Table 1) and having completed genetic testing were enrolled in the study, with their medical records reviewed retrospectively.Written informed consent was obtained from the patients who agreed to take part in the study.Demographic data (age, gender, age at diagnosis), clinical features (initial symptoms, presence of mucocutaneous skin lesions, polyp count and size in stomach, small intestine, and colon), family history, genetic analysis outcomes, and whether patients were diagnosed with cancer during follow-up were recorded.The median follow-up duration was 68.4 months.

Targeted Next-Generation Sequencing
The DNA was extracted from peripheral venous blood samples using the QIAamp® DNA Blood Mini Kit with the commercial QIAcube (Qiagen, Germany) following the manufacturer's protocol.Subsequently, the "Hereditary Cancer Susceptibility NGS panel" (Oncorisk Gene Panel) was performed, employing the "Celemics OncoRisk V3" NGS kit (Oncorisk, Celemics, South Korea).This panel encompasses 30 genes relevant to prediagnosed cases and was sequenced on the Nextseq 500 NGS platform (Illumina, CA, USA).Bioinformatic analysis of the obtained data was conducted using the Variant Analysis Platform for Genomics (SEQ analysis platform, Genomize, Türkiye).For each case, the STK11 gene achieved a minimum 94% coverage at a depth of 20× (Supplementary Table 1).Variants present in the ClinVar database classified as Benign or Likely Benign, as well as variants with an allelic frequency exceeding 5% in publicly available datasets like Exome Sequencing Project, 1000 Genomes Project, or Exome Aggregation Consortium, were excluded.Both coding and intronic regions (up to the kit's coverage) were included in the analysis.Variant pathogenicity was assessed following the 2015 American College of Medical Genetics and Genomics (ACMG) guidelines, 15 with inputs from the ClinVar database, Varsome (Saphetor, Switzerland), Franklin (Genoox, CA, USA) analysis platforms, and the SEQ platform.All identified variants underwent visual scrutiny using the Integrative Genomic Viewer software.

Multiplex Ligation-Dependent Probe Amplification Analysis for STK11
For patients in whom no mutations were detected using the targeted multigene NGS panel, the SALSA® MLPA® P101 STK11 probe mix (Microbiology Research Centre [MRC]-Holland, Amsterdam, Netherlands) was employed to screen for deletions and duplications within the STK11 gene.Although the lesions typically appeared before the initial complaint, they were not the primary reason for hospital admission.
Among the 20 patients, 18 underwent endoscopy and colonoscopy using the double-balloon enteroscopy technique.Gastric polyps were observed in 11 patients, small intestine polyps in 17 patients, and colon and rectum polyps in 13 patients (Table 2).
Dysplastic polyps were detected in 4 patients, and 3 patients developed breast, ovarian, and colorectal cancer.While 19 patients are actively being followed in our clinic, 1 patient died in 2019 due to complications related to ovarian cancer.
In cases where NGS analysis failed to detect pathogenic or likely pathogenic variants (6 patients), del/dup analysis using MLPA was conducted.Notably, exon 2 deletion was identified in 1 patient among these 3 cases.Additionally, a PTEN mutation (LRG_311: c.697C>T p.(Arg233*)) located in exon 7 was detected in a patient, prompting genetic counseling (Table 5).

Patient with LRG_319: c.598-8_601del
A 17-year-old patient presented with symptoms of nausea and vomiting.Physical examination revealed mucocutaneous lesions on the lower lip and buccal mucosa.The family history was unremarkable.Biopsy and endoscopy revealed the presence of duodenal polyps with hamartomatous features.
Patient with LRG_319: c.708_718del p.Asp237Glyfs*26 A 25-year-old male patient presented with rectal bleeding.The family history was unremarkable.Physical examination did not reveal any mucocutaneous polyps.Following the report of abdominal pain, the patient underwent endoscopic evaluation, revealing the presence of multiple hamartomatous polyps in the gastric, duodenal, and small intestinal regions.

Patient with LRG_319: c.146_147del
A 1-year-old male patient was brought to a pediatric outpatient clinic due to rectal prolapse.During the physical examination, a vermillion mucocutaneous lesion was noted.The patient's family history is notable, as the maternal grandmother had the same condition.Subsequent to complaints of abdominal pain, an endoscopic evaluation revealed multiple hamartomatous polyps in the gastric, duodenal, and small intestinal regions.Follow-up assessments disclosed the presence of multiple rectal polyps, with 2 of them exhibiting dysplasia.As a result, a colorectal resection was performed, involving the removal of a 20 cm segment extending from the colon to the sigmoid colon.Furthermore, an intraluminal mass and intussusception were identified in the distal jejunum.In addition to the STK11 mutation, the patient exhibited MUTYH mutation and CHEK2 variants (Table 5).Notably, the patient's age at diagnosis was significantly lower than both the study's mean age of 19 and the mean age reported in the literature.The early onset of symptoms and the implementation of prompt polypectomy could be linked to a truncated mutation.Nonetheless, it's worth considering that other mutations beyond STK11 might contribute to the formation of dysplastic polyps.

DISCUSSION
This study provides insights into PJS characteristics and genetics.Patient demographics and clinical manifestations align with existing literature, emphasizing the importance of PJS consideration in abdominal symptoms.Notably, mucocutaneous lesions were often overlooked, stressing the need for heightened awareness.Cancer emergence during follow-up confirmed PJS's malignancy risk.Genetic analysis demonstrated robust detection rates using the NGS panel, while cases with multiple gene alterations highlight the complexity of PJS's genetic landscape.Pathogenic or likely pathogenic variants were identified in the STK11 gene, including novel mutations.Our findings emphasize the need for comprehensive genetic analysis and early intervention strategies for effective PJS management.
7][18] These findings reinforce the relevance of PJS as a potential diagnosis in patients presenting with abdominal pain or intussusception.In particular, most patients presented with symptoms other than mucocutaneous lesions, highlighting the need for greater awareness among medical professionals and patients alike.
The distribution of polyps within our cohort mirrors studies from Germany and Taiwan, with significant occurrences in the gastric, small intestinal, and colorectal regions. 11,18Importantly, dysplastic polyps were identified in 20% of our patients, reinforcing the necessity of vigilant monitoring due to their inherent malignancy risk. 16,19ncer occurrence during follow-up, while lower in our study, underscores PJS's malignancy potential, 16,20,21 and was consistent with previous reports with breast, ovarian, and colorectal cancers emerging in our cohort. 18,22This variance might be attributed to the younger age of our  patient cohort, suggesting a need for prolonged follow-up to fully assess cancer incidence.
Genetic analysis was conducted using the Hereditary Cancer Susceptibility NGS panel, revealing pathogenic or likely pathogenic changes in PJS-related genes in a substantial portion of patients.The detection rate of 78.5% by NGS panel and 85.7% by NGS panel analysis and MLPA method collectively among patients meeting diagnostic criteria parallels align with previous reviews indicating its efficacy in uncovering pathogenic changes within STK11 gene. 6Our study, in line with previous genetic analyses, reinforces the utility of this approach. 23 addition, 3 of our patients (3/20) had alterations in LP/P class genes in more than 1 gene, as detected by panel testing.5][26][27][28][29] The complex etiology arising from mutations in more than 1 gene, leading to similar clinical presentations, adds complexity to the clinical interpretation.Notably, among these 3 cases carrying mutations in more than 1 cancer-related gene, the age of symptom onset is significantly earlier than in the remaining cases.However, the significance of this observation warrants validation through further comprehensive investigations.
Another aim of this study is the exploration of the phenotype-genotype relationship.We compared our cases of cancer and dysplastic polyps with existing literature to explore the phenotype-genotype relationship.In our cohort, 3 patients with mutations developed cancer; 2 with truncated mutations (breast and colorectal carcinoma), and 1 with a missense mutation (ovarian carcinoma).Several studies confirm elevated cancer risk linked to STK11 truncated variants. 30Amos et al 10 reported delayed GI symptoms and later first polypectomy in carriers of missense STK11 mutation compared to those with truncations.Also Salloch et al 11 found that individuals with truncated variants exhibited more polyps, earlier polypectomy, and higher cancer risk.Among patients with truncated mutations, we observed earlier symptom onset and heightened cancer risk, in line with earlier studies. 14,21This highlights the potential for tailored follow-up strategies to accommodate varying mutation profiles and optimize patient care carcinoma.
Regarding family patterns, our study aligns with past investigations, demonstrating variability in cancer development among family members sharing the same mutation. 10,12Additionally, domain XI mutations correlated with increased dysplasia risk, reinforcing the importance of genetic assessment for tailored patient management. 16 conclusion, this study provides valuable insights into the clinical and genetic features of PJS by integrating clinical and genetic insights.The findings underscore the critical importance of of genetic testing in

Figure 1 .
Figure 1.Schematic representation of the STK11 gene and mutations detected in the study.Modified from 9,10

Table 3 .
Clinical Features of the Cases

Table 4 .
Details of Identified STK11 Mutations in the Study PopulationThe American College of Medical Genetics and Genomics; dbSNP, The Single Nucleotide Polymorphism Database; GRCh38, Genome Reference Consortium Human Build 38; HGNC, HUGO Gene Nomenclature Committee; HGMD, Human Gene Mutation Database; HGVS, Human Genome Variation Society; HGVSc, the HGVS coding sequence name; HGVSp, the HGVS protein sequence name; Het, heterozygous; Hom, homozygous; LP, likely pathogenic; LRG, locus reference genomic; OMIM, Online Mendelian Inheritance in Man; P, pathogenic; VUS, variant of uncertain significance.

Table 4 .
Details of Identified STK11 Mutations in the Study Population (Continued)